A. Field of the Invention
The present invention relates generally to the detection of biological analytes such as cells, spores, or bacteria. More particularly, the present invention concern the immunomagnetic isolation and fluorescent detection of biological analytes that can be present in a wide range of concentrations and that may be located in a complex biological or environmental sample matrix.
B. Description of Related Art
The study, characterization and census of biological cells have always depended on the use of imaging tools that allow for the visualization of what the naked eye could not see directly. Since the first scientific observation of cells reported in 1665 by Robert Hooke (The Cell—A molecular approach; Geoffrey M. Cooper. ASM Press. 1997), the field of microscopy has enabled the field of cell biology. From the basic early bright field light microscopes to the most recent scanning electron microscopes, cell biologists exploited the increasing magnifications and resolution capabilities of these technologies. Today, the integration of digital imaging with these powerful microscopes has expanded the scope of this field even more. Microscopy can now be combined with powerful image processing techniques for the rapid and automated analysis of a wide variety of samples.
While the imaging capabilities of microscopes have evolved over centuries, sample preparation is usually still a discrete task performed prior to imaging. Usually a targeted element of interest in the sample (the nucleus of a cell, or the membrane of a cell, etc. . . . ) is stained or dyed in order to enable its visualization. There is no integrated apparatus that would perform all needed steps of an analysis such as the preparation of a sample, the imaging and characterization of a target cell, and then the regeneration of the imaging chamber after analysis of the sample. Microscope glass slides and cover slips are still widely used to accommodate samples due to their simplicity of use and low cost. Many lab-on-a-chip techniques use some form of microscopy (bright field or fluorescent microscopy) and imaging approach as a detector; however, there is often a lack of integration around the compact imaging chamber. In this respect, many lab-on-a-chip devices are still their infancy. Scanning electron microscope (SEM) instruments also lack sample preparation automation, and each sample is required to be individually mounted on a pin and coated with a metal prior to analysis. Microscopy systems therefore suffer from a lack of throughput. Sample preparation, positioning of the sample onto the focal plane and localization of the area of interest remain laborious processes.
The characterization of cells (size, shape) characterization of markers on the surface of cells can also be done by flow cytometry. Since the late 1970s, flow cytometry has enabled scientists to analyze a variety of cell types and offers numerous advantages over other cell-based techniques including: speed, preservation of cell viability and cellular functions, and simultaneous measurements of multiple cellular parameters.
The appeal of flow cytometry arises from the flexibility and sensitivity of fluorescence technology combined with the technique's high speed and powerful data integration capabilities. Flow cytometry currently is the gold standard method for cell sorting and analysis (Bioinformatics Market Research 2006 Report #06-030: “influencing brand preference in the flow cytometry market”). While Flow cytometers improve throughput of cell analysis, they are expensive and technically challenging to operate. Even the most affordable flow cytometer models are priced over $100,000, and more sophisticated models are priced at over $300,000. In addition, flow cytometers require well-trained operators and are very sensitive to shifts in their optical alignment. Consequently the overall purchasing and operating costs of these instruments make them inaccessible to many laboratories, particularly those located in resource-poor countries. Furthermore, flow systems are typically designed to take measurements from a single particle at a time, thus increasing the time required to collect images from multiple particles.